Metadata-Version: 2.1
Name: idemux
Version: 0.1.2
Summary: A Lexogen tool for demultiplexing and  index error correcting fastq files. Works with Lexogen i7, i5 and i1 barcodes.
Home-page: https://github.com/lexogen-tools/idemux
Author: Falko Hofmann, Michael Moldaschl, Andreas Tuerk
Author-email: falko.hofmann@lexogen.com, michael.moldaschl@lexogen.com, andreas.tuerk@lexogen.com
License: UNKNOWN
Description: ======================================
        idemux - inline barcode demultiplexing
        ======================================
        .. image:: https://badge.fury.io/py/idemux.svg
           :target: https://badge.fury.io/py/idemux
           :alt: Latest Version
        
        .. image:: https://travis-ci.org/lexogen-tools/idemux.svg?branch=master
           :target: https://travis-ci.org/lexogen-tools/idemux
        
        .. image:: https://coveralls.io/repos/github/Lexogen-Tools/idemux/badge.svg?branch=master&service=github
           :target: https://coveralls.io/github/Lexogen-Tools/idemux?branch=master
        
        
        Idemux is a command line tool designed to demultiplex paired-end FASTQ files from
        `QuantSeq-Pool <https://www.lexogen.com/quantseq-pool-sample-barcoded-3mrna-sequencing/>`_.
        
        Idemux can demultiplex based on i7, i5, and i1 inline barcodes. While this tool
        can generally be used to demultiplex any barcodes (as long as they are correctly supplied
        and in the fastq header), it performs best when used in combination with
        `Lexogen indices <https://www.lexogen.com/indexing/12nt-dual-indexing-kits/>`_, as it
        will correct common sequencing errors in the sequenced barcodes. This will allow you
        to retain more reads from your sequencing experiment while minimizing cross contamination.
        
        
        Idemux use is permitted under the following `licence <LICENCE>`_.
        
        **General usage:**
        ::
        
            idemux [-h] --r1 READ1 --r2 READ2 [--sample-sheet SAMPLE_SHEET]
                   --out OUTPUT_DIR [--i1-start I1_START] [--i5-rc] [-v]
        
        
        **Run idemux:**
        ::
        
            idemux --r1 read_1.fastq.gz --r2 read_2.fastq.gz --sample-sheet samples.csv --out /some/output/path --i1-start pos_in_read_2
        
        Features
        --------
        
        * FASTQ file demultiplexing based on i7, i5, and i1 barcodes.
        * Correction of barcode sequencing errors to maximize read yield (only works
          with `Lexogen 12 nt UDIs <https://www.lexogen.com/indexing/12nt-dual-indexing-kits/>`_
          that have been sequenced at least 8 nt).
        * Reverse complementation in case the i5 index has been sequenced as reverse complement.
        
        
        Getting started
        ---------------
        To get stated with demultiplexing you need to:
        
        1. `Install idemux <1. Installation_>`_
        2. `Prepare a sample sheet csv <2. Preparing the sample sheet_>`_
        3. `Run idemux <3. Running idemux_>`_
        
        1. Installation
        ===============
        
        Idemux is available on pypi. To install idemux via pip:
        
        ``$ pip install idemux``
        
        If you want to install idemux into a `virtual env <https://virtualenv.pypa.io/en/latest/>`_
        (always a good idea to avoid dependency conflicts), do the following:
        ::
        
            $ cd /path/you/want/it/installed/to
            # creates the venv
            $ virtualenv idemux
            # activates the venv, run 'deactivate' to deactivate
            $ source idemux/bin/activate
            $ pip install idemux
        
        
        Alternatively, you can clone this repository and install from there:
        ::
        
            $ cd /path/you/want/it/installed/to
            # creates the venv
            $ virtualenv idemux
            $ git clone https://github.com/Lexogen-Tools/idemux.git
            $ source idemux/bin/activate
            $ python setup.py install
        
        Additionally, Idemux will soon be available via bioconda!
        
        
        2. Preparing the sample sheet
        =============================
        In order to run idemux on your QuantSeq-Pool data, you first need to prepare a `csv file
        <https://en.wikipedia.org/wiki/Comma-separated_values>`_.
        We call this csv a sample sheet and it specifies which barcodes correspond to each
        sample.
        
        This is a necessity as the software needs to know which bins reads should be
        sorted into during demultiplexing. A sample sheet can easily be generated by filling in an
        excel spreadsheet and exporting it as csv.
        
        
        Example sample sheet (i7, i5, and i1 demuliplexing):
        ::
        
            sample_name,i7,i5,i1
            sample_0,AAAACATGCGTT,CCCCACTGAGTT,AAAACATGCGTT
            sample_1,AAAATCCCAGTT,CCCCTAAACGTT,AAAATCCCAGTT
            sample_2,GAAAATTTACGC,GCCCCTTTCAGA,GAAAATTTACGC
            sample_3,AAACTAACTGTC,CCCATCCATGTA,AAACTAACTGTC
        
        
        A sample sheet consists of 4 columns and always starts with the header illustrated
        above. 'Sample_name' values will be used as output file names, while the
        sequences specified in i7, i5, and i1 will be used for demultiplexing.
        
        Therefore, only unique, specific combinations of sample names and barcodes are
        allowed. This means using duplicated or ambiguous combinations will result in an error.
        However, idemux will do its best to tell you where the problem lies, if this happens.
        
        |
        
        **In brief, the rules are:**
        
        1. Sample names need to be unique.
        2. Barcode combinations need to be unique.
        3. i7 and/or i5 indices have to be used consistently within the csv file. i7 and/or i5 indices need to either be present for all samples or for none at all.
        4. In contrast to i7/i5 indices, i1 indices can be used for a subset of samples in the csv file.
        5. Absence of a barcode needs to be indicated by an empty field (no value between
           commas ``,,``).
        6. If your i5 has been sequenced as reverse complement, *do not* enter the reverse
           complement sequences in the sample sheet. Use the ``--i5-rc`` option!
        
        |
        
        See `below <Sample sheet examples_>`_ for more showcases of sample/barcode combinations that are *allowed* or
        *not allowed*.
        
        
        3. Running idemux
        =================
        Once you have installed the tool, you can run it by typing ``idemux`` in the terminal.
        
        Idemux accepts the following arguments:
        ::
        
            required arguments:
              --r1 READ1                   path to gzipped read 1 FASTQ file
              --r2 READ2                   path to gzipped read 2 FASTQ file
              --sample-sheet CSV           csv file describing sample names, and barcode combinations
              --out OUTPUT_DIR             where to write the output files
        
            optional arguments:
              --i5-rc                      when the i5 barcode has been sequenced as reverse complement.
                                           make sure to always use non-reverse complement sequences in the sample sheet
              --i1_start POS               start position of the i1 index (1-based) on read 2 (default: 11)
              -v, --version                show program's version number and exit
              -h, --help                   show help message and exit
        
        
        Example commands:
        ::
        
            # demultiplexes read 1 and 2 into the folder 'demux'
            idemux --r1 read_1.fastq.gz --r2 read_2.fastq.gz --sample-sheet samples.csv --out demux
        
            # demultiplexing assuming the i1 barcode starts at the first base
            idemux --r1 read_1.fastq.gz --r2 read_2.fastq.gz --sample-sheet samples.csv --out demux --i1_start 1
        
            # demultiplexing assuming i5 is present as reverse complement in the fastq header
            # if the i5 has been sequenced as reverse complement use this option and provide
            # the NON reverse complement sequences in the sample sheet.
            idemux --r1 read_1.fastq.gz --r2 read_2.fastq.gz --sample-sheet samples.csv --out demux
        
        After a successfully completed run, idemux will write a summary report to the output folder
        ('demultipexing_stats.tsv').
        
        Technicalities
        ---------------
        
        When you run idemux, the following will happen:
        
        * It will check if your sample sheet is okay. See `here <Sample sheet examples_>`_ for examples.
        
        * It will check the FASTQ header for barcodes and it expects them in the following format:
        
            single index (i7 or i5): @NB502007:379:HM7H2BGXF:1:11101:24585:1069 1:N:0:TCAGGTAANNTT
            
            where TCAGGTAANNTT is the sequence of the i7 or i5 index
        
            dual index (i7 and i5): @NB502007:379:HM7H2BGXF:1:11101:24585:1069 1:N:0:TCAGGTAANNTT+NANGGNNCNNNN
            
            where TCAGGTAANNTT is the sequence of the i7 index and NANGGNNCNNNN is the sequence of the i5 index.
        
        * Reads with incorrect i7,i5 or i1 index sequences which can be corrected by idemux will be written to the
          correct output file. However, the incorrect index sequence will not be replaced in the read header. This
          allows for additional processing of the incorrect sequences.
        * Reads that cannot be demultiplexed will be written to undetermined_R{1/2}.fastq.gz.
        
        * When you demultiplex based on i1 inline barcodes, a successfully recognized barcode
          sequence of 12 nt will be cut out and removed from read 2. This will leave
          you with the 10 nt UMI + the nucleotides that potentially follow the i1 barcode.
        
        This allows you to:
        
        1. Use other software, such as UMI_tools, to deal with the 10nt UMI, if desired.
        2. To demuliplex lanes where QuantSeq-Pool has been pooled with other libraries and read
           2 has been sequenced longer than the actual barcode.
        
        Help
        ------
        If you are demuliplexing a large number of samples (more than 500), you might encounter the
        following error:
        
        * ``OSError: [Errno 24] Too many open files``
        
        This error occurs because most OS have a limit on how many files can be opened and
        written to at the same time. In order to temporarily increase the limit on Linux run:
        ::
        
            # multiply your sample number*2 (as data is paired end)
            # then round to the next multiple of 1024
            $ ulimit -n the_number_above
        
        If you are looking for a permanent solution, you can change your ulimit values
        `this way <https://access.redhat.com/solutions/61334>`_.
        
        In case you experience any issues with this software please open an issue describing your
        problem. Make sure to post the version of the tool you are running (``-v, --version``)
        and your os.
        
        Sample sheet examples
        ---------------------
        *This is allowed:*
        ::
        
            # demultiplexing via full i7, i5, i1
            sample_name,i7,i5,i1
            sample_0,AAAACATGCGTT,CCCCACTGAGTT,AAAACATGCGTT
            sample_1,AAAATCCCAGTT,CCCCTAAACGTT,AAAATCCCAGTT
        
            # demultiplexing via full i7, i5 and sparse i1
            sample_name,i7,i5,i1
            sample_0,AAAACATGCGTT,CCCCACTGAGTT,AAAACATGCGTT
            sample_1,AAAATCCCAGTT,CCCCTAAACGTT,
        
            # demultiplexing via full i7, i5
            sample_name,i7,i5,i1
            sample_0,AAAACATGCGTT,CCCCACTGAGTT,
            sample_1,AAAATCCCAGTT,CCCCTAAACGTT,
        
            # demultiplexing via full i7, no i5 and sparse i1
            sample_name,i7,i5,i1
            sample_0,AAAACATGCGTT,,AAAACATGCGTT
            sample_1,AAAATCCCAGTT,,
        
            # demultiplexing via full i7 only
            sample_name,i7,i5,i1
            sample_0,AAAACATGCGTT,,
            sample_1,AAAATCCCAGTT,,
        
            # demultiplexing via full i5 and i1
            sample_name,i7,i5,i1
            sample_0,,CCCCACTGAGTT,AAAACATGCGTT
            sample_1,,CCCCTAAACGTT,AAAATCCCAGTT
        
            # demultiplexing via full i5 and sparse i1
            sample_name,i7,i5,i1
            sample_0,,CCCCACTGAGTT,AAAACATGCGTT
            sample_1,,CCCCTAAACGTT,
        
            # demultiplexing via full i5
            sample_name,i7,i5,i1
            sample_0,,CCCCACTGAGTT,
            sample_1,,CCCCTAAACGTT,
        
            # demultiplexing via full i1
            sample_name,i7,i5,i1
            sample_0,,,AAAACATGCGTT
            sample_1,,,AAAATCCCAGTT
        
        *This is not allowed:*
        ::
        
            # missing i1 column (or any other)
            sample_name,i7,i5,
            sample_0,AAAACATGCGTT,CCCCACTGAGTT
            sample_1,AAAATCCCAGTT,CCCCTAAACGTT
        
            # duplicated barcode combination
            sample_name,i7,i5,i1
            sample_0,AAAACATGCGTT,CCCCACTGAGTT,AAAACATGCGTT
            sample_1,AAAACATGCGTT,CCCCACTGAGTT,AAAACATGCGTT
        
            # duplicated sample names
            sample_name,i7,i5,i1
            sample_0,AAAACATGCGTT,CCCCACTGAGTT,AAAACATGCGTT
            sample_0,AAAATCCCAGTT,CCCCTAAACGTT,AAAATCCCAGTT
        
            # mixed, potentially ambiguous indexing (full i7 and sparse i5, i1)
            sample_name,i7,i5,i1
            sample_0,AAAACATGCGTT,CCCCACTGAGTT,AAAACATGCGTT
            sample_1,AAAATCCCAGTT,,AAAATCCCAGTT
            sample_2,GAAAATTTACGC,GCCCCTTTCAGA,GAAAATTTACGC
            sample_3,AAACTAACTGTC,,AAACTAACTGTC
        
            # mixed, potentially ambiguous indexing indexing (no i7, sparse i5 & i1)
            sample_name,i7,i5,i1
            sample_0,,CCCCACTGAGTT,
            sample_1,,,AAAATCCCAGTT
        
            # mixed, potentially ambiguous indexing indexing (sparse i7, full i5 & i1)
            sample_name,i7,i5,i1
            sample_0,,CCCCACTGAGTT,AAAACATGCGTT
            sample_1,AAAATCCCAGTT,CCCCTAAACGTT,AAAATCCCAGTT
            sample_2,,GCCCCTTTCAGA,GAAAATTTACGC
            sample_3,AAACTAACTGTC,CCCATCCATGTA,AAACTAACTGTC
        
            # missing comma separator
            sample_name,i7,i5,i1
            sample_0,AAAACATGCGTTCCCCACTGAGTT,AAAACATGCGTT
        
            # no barcodes
            sample_name,i7,i5,i1
            sample_0,,,
        
            # wrong column headers
            wrong_col_name,i7,i5,i1
            sample_0,AAAACATGCGTT,CCCCACTGAGTT,AAAACATGCGTT
        
        
        =======
        History
        =======
        
        
        0.1.2 (2020-08-21)
        ------------------
        
        * bumped version number
        
        
        0.1.1 (2020-08-21)
        ------------------
        
        * fixed rst files with linter
        * First release on test PyPI.
        
        0.1.0 (2020-08-21)
        ------------------
        
        * First building version.
        
Keywords: idemux
Platform: UNKNOWN
Classifier: Development Status :: 4 - Beta
Classifier: Intended Audience :: Science/Research
Classifier: Topic :: Scientific/Engineering :: Bio-Informatics
Classifier: Natural Language :: English
Classifier: Programming Language :: Python :: 3
Classifier: Programming Language :: Python :: 3.6
Classifier: Programming Language :: Python :: 3.7
Classifier: Programming Language :: Python :: 3.8
Requires-Python: >=3.6
Description-Content-Type: text/x-rst
Provides-Extra: docs
Provides-Extra: dev
